Use of Intramolecularly, Covalently Cross-Linked Proteins As Binding Partners In Immunoassays

ABSTRACT

The invention concerns the use of intramolecularly, covalently cross-linked proteins and covalently cross-linked reverse transcriptase from HIV as immunological binding partners in immunoassays. It also concerns immunological test procedures for detecting an analyte in a sample in which intramolecularly, covalently cross-linked proteins are used as binding partners, and it further concerns intramolecularly, covalently cross-linked reverse transcriptase from HIV and a method for producing this reverse transcriptase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/968,806, filed Jan. 3, 2008, which is a divisional of U.S. patentapplication Ser. No. 10/447,129, filed May 28, 2003, now U.S. Pat. No.7,351,799, which is a continuation of international applicationPCT/EP01/13780, filed Nov. 27, 2001, which claims priority to Germanapplication DE 10059720.3, filed Nov. 30, 2000.

FIELD OF THE INVENTION

The present invention concerns the use of intramolecularly, covalentlycross-linked proteins, and in particular, the use of covalentlycross-linked reverse transcriptase (RT) from HIV as immunologicalbinding partners in immunoassays, and to immunological test proceduresfor detecting an analyte in a sample in which intramolecularly,covalently cross-linked proteins are used as binding partners. It alsoconcerns intramolecularly, covalently cross-linked reverse transcriptasefrom HIV and a method for producing this reverse transcriptase.

BACKGROUND OF THE INVENTION

The use of proteins as binding partners for the detection of analytes inimmuno-diagnostic test procedures has been known for a long time. In allconventional immunoassays, the sample is incubated with one or morebinding partners that are specific for the analyte. The binding partneror binding partners bind(s) specifically to the analyte to be detected.In the case of an antibody test, for example, in the case of an HCVinfection, the sample to be examined is, for example, contacted with anHCV antigen which specifically binds the anti-HCV antibody to bedetected. In an antigen test, for example, for detecting the tumourmarker prostate-specific antigen (PSA), the sample is contacted withantibodies which specifically bind the PSA in the sample.

Subsequently the analyte is detected in all immunoassays. This can, forexample, be carried out by binding and subsequently detecting anotherbinding partner provided with a detectable label which binds to thecomplex consisting of analyte and immunological binding partner.

In general the immunoassays are carried out in a heterogeneous orhomogeneous test format. The heterogeneous test formats are frequentlycarried out as sandwich or bridge tests. Competitive methods are alsowell known in which either the analyte or the specific binding partneris displaced from the complex of analyte and specific binding partnerby, for example, adding a labelled analyte analogue.

In all immunological test methods, it is important that the reactantsused as the specific immunological binding partners are present in astable form and that they are not destroyed, for example, byunfavourable storage conditions. This risk can occur in particular whenthe proteins used as specific binding partners are composed of severalsubunits. The subunits can be held together covalently, for example, bymeans of disulfide bridges, or non-covalently, for example, by means ofhydrogen bridges, opposite charges, and/or hydrophobic interactions.

In some cases, the materials required for the immunological test maybecome unstable and denature under the storage conditions (for example,as a liquid reagent) in the working solutions prepared for the test orduring the immunological reaction itself. As a result, the tertiary andthe quaternary structure of the protein may be changed in such a mannerthat the substance can no longer be used in the immunoassay.

The subunit components of the proteins used as a specific binding pairmay separate under unfavourable conditions. This dissociation ofsubunits may, for example, be caused by the reduction of disulfidebridges by common buffer additives such as DTT in the case of naturalcovalent bridges.

However, the risk of dissociation is even higher in the case ofnon-covalently linked subunits of a protein which are held together bycharges or hydrophobic interactions. The subunits of such proteins canbe very easily dissociated even by common buffer additives such assalts, detergents, or unfavourable variations in pH and temperature. Anindividual and hence unprotected subunit is thus also susceptible todenaturation. This may lead to major changes in the tertiary structureof the protein or of the individual subunit. This also means that theimmunological properties such as the accessibility of important epitopesis changed to such an extent that the protein used as a binding partnerin the immunoassay is no longer recognized immunologically and is henceno longer specifically bound.

Another risk of subunit dissociation is that subunits provided withdifferent labelling groups may re-associate due to the adjustment of thechemical equilibrium. If in a specific case, a protein composed of twosubunits for use in an antibody test in a bridge test format isderivatized in order to be used as a universal solid phase and, on theother hand, the same protein is also used as a signal-generatingcomponent and for this purpose is coupled to a label (e.g., an enzyme,fluorescent label, or chemilumineseent label), the following may happen:a calibration curve which is initially generated with positive samples(samples which contain the analyte) becomes flatter as time progresses.The signals for negative samples (blank values) increase andincreasingly approximate the values for the upper positive samples sothat it is no longer possible to differentiate between analyte-free andanalyte-containing samples.

A method for chemically modifying enzymes by reaction with quinones isdescribed in German Patent Application DE 26 15 349. These modificationsincrease the stability which results in an improved enzyme activity. Itis mentioned that the enzyme molecules can be cross-linked to oneanother, i.e., intermolecularly and also intramolecularly. In this case,the preservation of immunoreactive epitopes is irrelevant. The use ofenzymes modified with quinones in immunodiagnostic methods is notdescribed.

Debyser and De Clercq (Protein Science 5, p. 278-286, 1996) describe thecross-linking of the two subunits of HIV-1 reverse transcriptase bymeans of dimethyl suberimidate which cross-links lysine side chains. Thepurpose of the cross-linking is to examine the dimerization of the twoRT subunits. Only the dimeric RT is enzymatically active. The twosubunits are covalently cross-linked in the presence of variousinhibitors. RT molecules and multimers that are more or less strongcross-linked depending on the effectiveness of the inhibitor are formedafter the chemical cross-linking reaction. The effect of thecross-linking on immunologically relevant epitopes or the use ofcross-linked molecules in immunoassays is unimportant.

The use of intermolecularly cross-linked immunoglobulins in immunoassaysis disclosed in EP-A-0 331 068. This means that several immunoglobulinmolecules or fragments thereof are covalently linked together. Themultimers of antibodies and fragments thereof are used as aninterference-reducing reagent. The cross-linked immunoglobulins andfragments thereof are intended to eliminate interfering factors of humanserum that are directed towards immunoglobulins.

The cross-linked proteins described in the prior art, which are composedof several subunits under natural conditions, are unsuitable or of onlylimited suitability for use as antigens or immunological bindingpartners since, in general, intermolecular multimers consisting ofseveral protein molecules are formed. These multimers are of onlylimited use for immunoassays since they usually do not have a definedsize. Hence the multimers have a random distribution of sizes, i.e.,mono-, di-, tri-, tetramers, etc. are present together in one mixture.The undefined cross-linking may mask the epitopes. Consequently a sampleantibody to be detected may not be able to bind to the masked epitope ofthe antigen, and hence a false negative result is obtained.

Another problem with using multimers as immunological binding partnersis the fact that there is an increased risk that interfering factorspresent in the sample may bind unspecifically to the multimericproteins. Interfering factors such as rheumatoid factors often haveseveral binding sites of low affinity. If multimeric proteins are thenused as immunological binding partners, this may have the effect thatespecially the interfering factors find many targets, i.e., bindingsites on the multimeric proteins. This may lead to false positive testresults, and the overall specificity of the immunoassay is greatlyreduced.

SUMMARY OF THE INVENTION

Hence the object was to provide proteins with an improved stabilitywhich can be used in immunoassays as binding partners. The proteinsimproved in this manner should have good epitope accessibility, and thespecificity of the immunological test procedure in which the proteinsare used should be maintained.

The object is achieved by the invention described in the independentclaims. The dependent claims represent preferred embodiments.

It surprisingly turned out that proteins that are almost exclusivelyintramolecularly cross-linked can be produced without loss of theirimmunological properties, and these proteins can be used in anadvantageous manner in immunological test procedures as immunologicalbinding partners. The stability problems that occur when the proteinsare not cross-linked are thus substantially avoided. Hence the inventionconcerns the use of intramolecularly, covalently cross-linked proteinsas immunological binding partners in immunological test procedures.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the analysis by gel permeation chromatography of themolecular weights of RT obtained alter cross-linking.

DETAILED DESCRIPTION OF THE INVENTION

All proteins required for immunological test procedures that arefamiliar to a person skilled in the art can be used as the proteins. Allpolypeptides can be used which could, as a result of their folding,i.e., their tertiary or quaternary stricture, have a tendency to unfold,to denature, or to dissociate into various subunits under the conditionsof an immunoassay. When such a structural change occurs, there is a riskthat immunologically important epitopes are changed in such a mannerthat, for example, they are no longer specifically bound by antibodies.In the worst case, this means that an immunological test result isnegative, i.e., it does not indicate the presence of the antibody to bedetected because the proteins used as binding partners are denatured.These disadvantages are substantially avoided by the use ofintramolecularly, covalently cross-linked proteins according to theinvention.

In particular, those intramolecularly, covalently cross-linked proteinsare used which are naturally composed of several subunits. DNA or RNApolymerases, particularly the reverse transcriptase from HIV, andespecially preferably the reverse transcriptase from HIV-1 arepreferably used.

The proteins can be from any desired source. The proteins to becross-linked can be isolated from their natural source such as anorganism or virus. However, the use of recombinant proteins produced bygenetic engineering is preferred. A recombinant purified RT isespecially preferably used which is expressed by an expression clone asdescribed, for example, in Müller et al., J. Biol. Chem.264/24:13975-13978 (1989).

With the intramolecularly, covalently cross-linked proteins, it isimportant that the epitopes that are important for immunologicalrecognition are not changed by the cross-linking or only so slightlythat the other immunological binding partner in the test recognizes andspecifically binds the cross-linked protein just as well as theuncrosslinked protein. Hence the cross-linking should not generate anyimmunologically relevant artefacts that could falsify the test result.

The “protein” refers to all polypeptides which are composed of at leastabout 50 amino acids, preferably of at least 100 amino acids. The termprotein also includes modified proteins such as proteins that are linkedwith sugar residues, sialic acids, or lipid structures.

The term “intramolecularly, covalently cross-linked” refers to proteinswhose polypeptide chain has been linked together by chemicalmodification in such a manner that it can no longer unfold, i.e., it canno longer lose its tertiary structure and thus the accessibility ofimportant epitopes. In the case of a protein which is composed ofseveral subunits, the intramolecular, covalent cross-linking maintainsthe tertiary as well as the quaternary structure. The modificationprevents the various polypeptide chains from diffusing away from oneanother.

It is important that the covalent linkage only occurs within a proteinmolecule. In the case of proteins which are only composed of onepolypeptide chain and thus of only one subunit under natural conditions,at least two sites within a polypeptide chain are linked. Hence nooligomers consisting of several proteins are formed by theintramolecular, covalent cross-linking. Such oligomers are also referredto in the following as polymers or multimers.

Hence the molecular weight of the intramolecularly, covalentlycross-linked proteins is only increased if the chemical linker iscovalently bound to the protein, and hence the total mass is slightlyincreased.

In the case of proteins which are composed of several subunits, thelinkage according to the invention only occurs between those subunitswhich also naturally form an intact protein molecule. This means thatthe size and the molecular weight of the intramolecularly, covalentlycross-linked protein according to the invention is only slightlyincreased by the cross-linking chemical substance. Linkages withinseveral protein molecules are virtually excluded so that oligomers oreven polymers of the proteins form.

According to the invention, the cross-linked proteins can be providedwith other modification groups before or after the cross-linking which,for example, are required for their application as labelled antigens orin order to bind the cross-linked proteins to a solid phase. Forexample, they can be linked with biotin, streptavidin, or withsignal-generating labelling groups such as enzymes, fluorescent groups,or chemiluminescent groups. Such modifications are familiar to a personskilled in the art. These modifications should not change theimmunological properties of the intramolecularly cross-linked proteinsaccording to the invention, or only to such an extent that a recognitionby the specific binding partner in the immunoassay is still ensured.

The almost exclusive intramolecular linkage of the proteins can, forexample, be detected by means of SDS polyacrylamide gel electrophoresis(SDS-PAGE) with subsequent Coomassie blue staining, especially in thecase of proteins having a quaternary structure. After the proteincross-linking according to the invention, it should not be possible todetect any molecular weights with the naked eye that are larger thanthat of the natural molecular weight of the protein in an SDS-PAGE gel.If, for example, a miniaturized commercial SDS-polyacrylamide gradientgel of 8 to 25% polyacrylamide (PHAST system from Pharmacia) is used,the amount of protein applied per lane is about 500 ng. With this amountof protein, molecular weights that are larger than that of the naturalmolecular weight cannot be detected in this system with the naked eyeaccording to the invention. In the case of proteins which naturally haveseveral subunits, i.e., several polypeptide chains, the molecular weightof a band after cross-linking should not exceed the sum of the molecularweights of the subunits. Protein bands on the gel which have a molecularweight corresponding to the sum of the molecular weights of the subunitsmay be regarded as a test for a successful intramolecular cross-linkingof a protein having a quaternary structure. Hence SDS-PAGE can be usedto establish the successful intramolecular cross-linking of a proteinconsisting of several subunits and the absence of multimers.

Another method for detecting the absence of multimers is by means of gelpermeation chromatography, also referred to as gel exclusionchromatography, which can, for example, be carried out using acommercial HPLC apparatus. Protein complexes which have a molecularweight corresponding to a multimer of the individual protein are elutedsubstantially earlier than proteins that are present singly. Accordingto the invention, only a low percentage of such multimers should bepresent. If one measures the integral of a HPLC chromatogram, this meansthat no more than about 5% multimers should be present relative to theeluted peak (integral) of the protein according to the invention whichis only cross-linked intramolecularly.

“Immunological binding partners” refers to all molecules which canspecifically bind to other molecules under the conditions of animmunoassay. In particular, immunological binding partners should beable to specifically bind the analyte or a substance bound to theanalyte. A classical constellation is the specific binding of anantibody to an antigen, for example, the binding of an anti-PSA antibodyto PSA. Antibodies and antigens are immunological binding partners.According to the invention, intramolecularly, covalently cross-linkedproteins are used as immunological binding partners in immunoassays.

Antigens are preferably used as immunological binding partners when itis intended to detect an antibody directed against these antigens. Inthis case, the detection of anti-HIV RT antibodies by means of HIVreverse transcriptase that is cross-linked according to the invention ispreferred and is described in a later section.

The invention also concerns an immunological test procedure fordetecting an analyte in a sample. The method is characterized in that anintramolecularly, covalently cross-linked protein is used as theimmunological binding partner. It has turned out that intramolecularly,covalently cross-linked proteins, and in particular, those that arenaturally composed of several subunits, are considerably more stablethan uncrosslinked proteins under the conditions of an immunoassay.

The various formats and embodiments of immunoassays as well as thevarious detection methods, such as by means of enzymatic reactions,fluorescent substances, or chemiluminescent substances, are familiar toa person skilled in the art and do not therefore need to be speciallyelucidated here. A heterogeneous test format is preferred according tothe invention in which the solid phase is separated from the liquidphase after completion of the immunological reaction.

The method is preferably an immunoassay for diagnosing HIV infections.If a patient has an HIV infection, this can be detected on the basis ofantibodies that have been formed against certain antigens of the virusin a blood, serum or plasma sample. It is often also possible to detectthe viral antigens of the HIV itself such as the p24 antigen of HIV-1.This requires the use of specific antibodies directed against the HIVantigen, in this case, against p24.

The detection of an HIV infection in a sample is often carried out as acombined antigen and antibody detection test. Such tests are referred toas COMBI-TESTs. Such a COMBI-TEST is described in WO 98/40744. In thiscase, HIV antigens, i.e., the p24 antigen of HIV-1 or HIV-1 subtype Oand the corresponding p26 antigen of HIV-2, are detected by means ofspecific antibodies as well as antibodies directed against HIV andspecifically against envelope proteins (env) of the pathogen such asgp160, gp120, and gp41 of HIV-1 and gp140, gp110, and gp36 of HIV-2. Inaddition, antibodies against HIV-RT are also detected in the combitestaccording to WO 98/40744. For this purpose, HIV-1 reverse transcriptaseproduced recombinantly is used as an immunological binding partnerwhich, however, is not intramolecularly covalently cross-linked.

According to the invention, intramolecularly, covalently cross-linked RTfrom HIV, in particular RT from HIV-1, is preferably used in aCOMBI-TEST to detect an HIV infection in a sample.

Another subject matter of the invention is intramolecularly, covalentlycross-linked reverse transcriptase from HIV, an enzyme which isnaturally present in two subunits. The HIV-RT is present as aheterodimer under natural conditions. HIV-1 RT is composed of 1 subunitof 51 kDa and one subunit of 66 kDa. The recombinant form can, forexample, be obtained from expression clones (for example, from Müller etal., J. Biol. Chem. 264/24, p. 13975-13978, 1989). Due to a degree ofhomology of about 60% and even of 100% in some sections at the aminoacid level, the HIV-1 RT can in general also be used to detectantibodies directed against HIV-2 RT. The term “HIV” includes HIV-1,HIV-2, and all subtypes and subgroups of the virus such as the HIV-1subtype O. HIV-1 RT in an intramolecularly, covalently cross-linked formis preferred.

It has surprisingly turned out that the intramolecularly, covalentlycross-linked HIV RT is considerably more stable than the uncrosslinkedform under the conditions of the immunoassay. The RT according to theinvention was considerably better than the uncrosslinked form andwithstood temperature stress to which it is, for example, exposed onlonger or improper storage or under assay conditions. Theintramolecularly, covalently cross-linked RT according to the inventionis characterized in that the two subunits are covalently linkedtogether, but there is no intermolecular cross-linking of severalmolecules. It can, for example, be demonstrated that no oligomers ofseveral RT molecules are present by using gel exclusion chromatographyor SDS-PAGE as already elucidated.

Homo- and heterobifunctional linkers are preferably used ascross-linking reagents. In particular, the following are preferably usedto intramolecularly cross-link the RT: MHS(3-maleimidobenzoyl-N-hydroxysuccinimide ester), EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), DSS(disuccinimidylsuberate), HSAB (N-hydroxysuccinimidyl-4-azidobenzoate),and sulfo-SANPAH(sulfosuccinimidyl-6(4′-amido-2′-nitrophenylamido)hexanoate). As alreadyelucidated, it is important that the chemical reaction of thecross-linking linker only results in an intramolecular cross-linking ofthe protein or of the two RT subunits but not a cross-linking betweenseveral RT molecules. In addition, it is important that noimmunologically relevant epitopes are destroyed by the chemicalreaction.

Another subject matter of the invention is a method for producingintramolecularly cross-linked HIV reverse transcriptase. The methodcomprises the steps:

-   -   providing RT in a dissolved form,    -   optionally reacting the RT with a blocking reagent for SH        groups,    -   dialysing the mixture against aqueous buffer,    -   reacting the activated RT with one of the cross-linking reagents        MHS (3-maleimidobenzoyl-N-hydroxysuccinimide ester), EDC        (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), DSS        (disuccinimidylsuberate), HSAB        (N-hydroxysuccinimidyl-4-azidobenzoate), and sulfo-SANPAH        (sulfosuccinimidyl-6(4′-amido-2′-nitrophenylamido)hexanoate),    -   optionally stopping the reaction,    -   separating the excess reactants from the reaction product by        dialysis, and    -   optionally exposing the dialysed reaction product to UV light.

The preferred stoichiometry of RT to cross-linking reagent is about 1:1to 1:20. The ratios of the reactants are selected such that nooligomerization or only a negligible oligomerization occurs betweenseveral RT molecules.

The invention is further elucidated by the following examples.

EXAMPLES Example 1 Intramolecular Cross-Linking of HIV-1 ReverseTranscriptase

a) Cross-Linking with MHS

HIV-1 reverse transcriptase (10 mg/ml) was dissolved in 50 mMdiethanolamine, pH 8.8, 25 mM NaCl, 1 M DTT, and 1 mM EDTA. The pH wasadjusted to 6.4 by adding a 1 M KH₂PO₄ solution.

The mixture was adjusted to 5 mM NMM by adding an appropriate aliquot ofa 1 M solution of NMM (N-methylmaleinimide) in DMSO and subsequentlyincubated for 60 min at 25° C. while stirring. It was subsequentlydialysed against 50 mM diethanolamine, pH 8.8, 25 mM NaCl.

The pH was then adjusted to pH 7.0 by adding a 1 M KH₂PO₄ solution. Astock solution of MHS (3-maleimidobenzoyl-N-hydroxysuccinimide ester)was prepared in DMSO (5 mg/ml). A quantity of this solutioncorresponding to an initial stoichiometry of 1:8 (mol reversetranscriptase/mol MHS) was added to the mixture which was then incubatedfor a further 60 min at 25° C. while stirring. The reaction wasterminated by adding lysine to the reaction mixture at a finalconcentration of 10 mM and incubating for a further 30 min. Excessreactants were separated by dialysis against 10 mM potassium phosphatebuffer, pH 6.0, 50 mM NaCl, 1 mM EDTA.

After dialysis the pH was adjusted to 7.4 by adding an aliquot of a 1 MK₂HPO₄ solution. The mixture was incubated for a further 4 h at 25° C.while stirring, before adding cysteine to a final concentration of 2 mM.After a further 30 min incubation, the reaction was terminated by addingNMM (final concentration 5 mM). The mixture was dialysed against 50 mMdiethanolamine, pH 8.8, 25 mM NaCl.

b) Cross-Linking with EDC

HIV-1 reverse transcriptase (10 mg/ml) was dissolved in 50 mMdiethanolamine, pH 8.8, 25 nM NaCl, 1 mM DTT, and 1 mM EDTA. The pH wasadjusted to 6.4 by adding a 1 M KH₂PO₄ solution.

The mixture was made up to 5 mM NMM by adding an appropriate aliquot ofa 1 M solution of NMM (N-methylmaleinimide) in DMSO and subsequentlyincubated for 60 min at 25° C. while stirring. It was subsequentlydialysed against 10 mM potassium phosphate buffer, pH 7.0, 50 mM NaCl.

A stock solution of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)was prepared in DMSO (2 mg/ml). A quantity of this solutioncorresponding to an initial stoichiometry of 1:10 (mol reversetranscriptase/mol EDC) was added to the mixture, which was thenincubated for a further 60 min at 25° C. while stirring. Excessreactants were separated by dialysis against 25 mM potassium phosphatebuffer, pH 7.0, 50 mM NaCl.

c) Cross-Linking with DSS

HIV-1 reverse transcriptase (10 mg/ml) was dissolved in 50 mMdiethanolamine, pH 8.8, 25 mM NaCl, 1 mM DTT, and 1 mM EDTA. The pH wasadjusted to 6.4 by adding a 1 M KH₂PO₄ solution.

The mixture was made up to 5 mM NMM by adding an appropriate aliquot ofa 1 M solution of NMM (N-methylmaleinimide) in DMSO and subsequentlyincubated for 60 min at 25° C. while stirring. It was subsequentlydialysed against 10 mM potassium phosphate buffer, pH 8.0, 25 mM NaCl.

A stock solution of DSS (disuccinimidyl suberate) was prepared in DMSO(2 mg/ml). A quantity of this solution corresponding to an initialstoichiometry of 1:10 (mol reverse transcriptase/mol DSS) was added tothe mixture, which was then incubated for a further 60 min at 25° C.while stirring. Excess reactants were separated by dialysis against 25mM potassium phosphate buffer, pH 7.0, 50 mM NaCl.

d) Cross-Linking with HSAB

HIV-1 reverse transcriptase (10 mg/ml) was dissolved in 50 mMdiethanolamine, pH 8.8, 25 mM NaCl, 1 mM DTT, and 1 mM EDTA. The pH wasadjusted to 6.4 by adding a 1 M KH₂PO₄ solution.

The mixture was made up to 5 mM NMM by adding an appropriate aliquot ofa 1 M solution of NMM (N-methylmaleinimide) in DMSO and subsequentlyincubated for 60 min at 25° C. while stirring. It was subsequentlydialysed against 10 mM potassium phosphate buffer, pH 8.0, 25 mM NaCl.

A stock solution of HSAB (N-hydroxysuccinimidyl-4-azidobenzoate) wasprepared in DMSO (2 mg/ml). A quantity of this solution corresponding toan initial stoichiometry of 1:5 (mol reverse transcriptase/mol HSAB) wasadded to the mixture, which was then incubated for a further 60 min at25° C. while stirring. Excess reactants were separated by dialysisagainst 25 mM potassium phosphate buffer, pH 7.0, 50 mM NaCl.

The mixture was subsequently irradiated for 7 min with a UV lamp.

e) Cross-Linking with Sulfo-SANPAH

HIV-1 reverse transcriptase (10 mg/ml) was dissolved in 50 mMdiethanolamine, pH 8.8, 25 mM NaCl, 1 mM DTT, and 1 mM EDTA. The pH wasadjusted to 6.4 by adding a 1 M KH₂PO₄ solution.

The mixture was made up to 5 mM NMM by adding an appropriate aliquot ofa 1 M solution of NMM (N-methylmaleinimide) in DMSO and subsequentlyincubated for 60 min at 25° C. while stirring. It was subsequentlydialysed against 10 mM potassium phosphate buffer, pH 8.0, 25 mM NaCl.

A stock solution of sulfo-SANPAH(sulfosuccinimidyl-6(4′-amido-2′-nitrophenyl-amido)hexanoate) wasprepared in DMSO (4 mg/ml). A quantity of this solution corresponding toan initial stoichiometry of 1:5 (mol reverse transcriptase/molsulfo-SANPAH) was added to the mixture, which was then incubated for afurther 60 min at 25° C. while stirring. Excess reactants were separatedby dialysis against 25 mM potassium phosphate buffer, pH 7.0, 50 mMNaCl.

The mixture was subsequently irradiated for 7 min with a UV lamp.

Example 2 Detection of the Exclusive Intramolecular Cross-Linking ofHIV-1 Reverse Transcriptase a) SDS Gel Electrophoresis

Aliquots of the intramolecularly cross-linked HIV-1 reversetranscriptase were analysed by polyacrylamide gel electrophoresis in thepresence of SDS on a PHAST gel apparatus (Pharmacia) according to astandard protocol of the manufacturer.

The non-cross-linked control only has bands with molecular weights of 66kD and 51 kD which correspond to the subunits of the reversetranscriptase. Intramolecularly cross-linked reverse transcriptaseexhibits bands with molecular weights of 110-120 kD, which demonstratesa successful cross-linking between subunits. Larger protein complexesare not detectable, i.e., an intramolecular linkage of several moleculesof reverse transcriptase does not occur with the cross-linking methodaccording to the invention.

b) Analytical Gel Permeation Chromatography

An aliquot of the intramolecularly cross-linked HIV-1 reversetranscriptase was analysed by gel permeation chromatography on a TSK3000 column (Toso Haas) using a commercial HPLC apparatus according to astandard protocol of the manufacturer.

The intramolecularly cross-linked reverse transcriptase elutes from thecolumn with a retention time that corresponds to globular proteinshaving molecular weights of 100-130 kD (in this case 7.5 min). Largerprotein complexes which would have a shorter retention time in thechromatogram are not detectable, i.e., the cross-linking methodaccording to the invention does not result in an intermolecularcross-linking of several molecules of reverse transcriptase to formoligomeric or polymeric structures. The chromatogram is shown in FIG. 1.

Example 3 Derivatization of Intramolecularly Cross-Linked HIV-1 ReverseTranscriptase with a Biotin Label

Intramolecularly cross-linked HIV-1 reverse transcriptase (seeexample 1) was present in diethanolamine or potassium phosphate buffer.The uncrosslinked RT was treated as a comparison with N-methylmaleimideand dialysed against diethanolamine. If necessary, the pH was adjustedto 8.6-8.8 in all RT mixtures by adding NaOH. A stock of biotin-DDS(biotinyl-diaminodioxaoctane-disuccinimidyl suberate) was prepared inDMSO (6 mg/ml). A quantity of this solution corresponding to an initialstoichiometry of 1:4 (mol reverse transcriptase/mol biotin-DDS) wasadded to the mixture, which was then incubated for a further 60 min at25° C. while stirring. The reaction was terminated by adding lysine tothe reaction mixture to a final concentration of 10 mM and incubatingfor a further 30 min. Excess reactants were separated by dialysisagainst 50 mM diethanolamine, pH 8.8, 25 mM NaCl.

Example 4 Stability Check in a Function Test

An immunoassay was carried out on an ELECSYS® analyzer from RocheDiagnostics GmbH, Mannheim, to examine the stability of the HIV-1reverse transcriptase. In addition to a negative control (NC) whichcontained no anti-RT antibodies and a positive control (PC) whichcontained anti-RT antibodies, two HIV-positive human sera with anti-RTreactivity were measured.

45 μl sample was incubated together with 55 μl Reagent 1 (biotinylatedRT) and 55 μl Reagent 2 (ruthenium-labelled RT) for 9 min at 37° C.Subsequently streptavidin-coated magnetic beads were added, and themixture was incubated for a further 9 min. Afterwards the beads werecaptured by a magnet, and the electrochemiluminescence signal wasquantified.

In order to compare the stability of biotinylated reverse transcriptasein the cross-linked form according to the invention and in anuncrosslinked form, Reagent 1 (biotinylated RT) was incubated for 18hours at 42° C. as described below before carrying out the test. Reagent1, which was prepared at the same time and stored at 4° C., served as areference. All other reagents were freshly prepared for the experiments.

The evaluation was based on the dynamic range of the signal which meansthat one determines the quotients of the signal and the respectivenegative control. The larger the value for signal dynamics the greateris the differentiation between HIV antibody-positive and negativesamples. Hence a large dynamic range of the signal is desirable. Therelation between the respective values was used to compare stressed RTand unstressed RT. The results are shown in Table 1.

TABLE 1 Unstressed Stress 18 h 42° C. Comparison stressed/unstressedSignal Signal Signal dynamic dynamic dynamic Samples Counts range Countsrange Counts range Recombinant HIV-1-RT-Bi(DDS), non-cross-linkedNegative control 1763 1.0 1049 1.0 60% 100% Positive control 16848 9.61480 1.4  9% 15% HIV serum 1 6209 3.5 1054 1.0 17% 29% HIV serum 2 51622.9 917 0.9 18% 30% HIV serum 3 5832 3.3 1150 1.1 20% 33% HIV serum 4111444 63.2 6267 6.0  6% 9% Recombinant HIV-1-RT (MHS)-Bi(DDS),cross-linked according to the invention Negative control 1304 1.0 12061.0 92% 100% Positive control 73335 56.2 77611 64.4 106%  114% HIV serum5 8476 6.5 7092 5.9 84% 90% HIV serum 6 14504 11.1 10615 8.8 73% 79% HIVscrum 7 69459 53.3 59347 49.2 85% 92% HIV serum 8 168674 129.4 168304139.6 100%  108%

Even after stress for several hours at an elevated temperature, thedynamic range of the signal in the immunoassay using RT cross-linkedaccording to the invention was still at least 79% and preferably atleast 90% compared to unstressed RT, whereas the dynamic range based onthe negative control was at most about 30% and sometimes considerablyless than 30% or even below 20% in the case of uncrosslinked RT. Whenusing uncrosslinked RT, the signal dropped to the level of negativesera, whereas the cross-linked RT according to the invention retains itsimmunological function. Hence the RT epitopes recognized by the sampleantibody are substantially preserved despite the thermal stress. Thismeans that the cross-linked RT according to the invention isconsiderably more stable than uncrosslinked RT.

1. A method for detecting an antibody in a sample comprising the stepsof: (a) combining the sample with an intramolecularly, covalentlycross-linked protein antigen in the absence of detectable intermolecularmultimers of the antigen, wherein the antigen specifically binds withthe antibody or with a substance bound to the analyte to form a complex,(b) adding to the combination formed in step (a) a binding partnerprovided with a label that combines with the complex formed in step (a)to produce a detectable signal, and (c) determining the signal producedin step (b) as a measure of the analyte in the sample.
 2. The method ofclaim 1 wherein the protein antigen is selected from the groupconsisting of DNA and RNA polymerases.
 3. The method of claim 1 whereinthe protein antigen is prostate specific antigen.
 4. A method fordetecting an antibody in a sample comprising the steps of: (a)contacting the sample with a composition comprising an intramolecularly,covalently cross-linked protein antigen, wherein said compositioncomprises less than about 5% of total intermolecular multimers of theprotein antigen relative to the eluted peak intramolecularly, covalentlycross-linked protein antigen as determined in a gel permeationchromatograph, wherein the intramolecularly, covalently cross-linkedprotein antigen specifically binds with the antibody or with a substancebound to the antibody to form a complex, (b) adding to the combinationformed in step (a) a binding partner provided with a label that combineswith the complex formed in step (a) to produce a detectable signal, and(c) determining the signal produced in step (b) as a measure of theanalyte in the sample.
 5. The method of claim 4 wherein intermolecularmultimers of the protein antigen are undetectable using SDS-PAGEanalysis when 500 ng of said composition is loaded per lane.
 6. Themethod of claim 4 wherein intermolecular multimers of the proteinantigen are undetectable using gel permeation chromatography analysis.7. The method of claim 4 wherein the protein is selected from the groupconsisting of DNA and RNA polymerases.
 8. The method of claim 4 whereinthe protein antigen is cross-linked using a cross-linking agent selectedform the group consisting of MHS(3-maleimidobenzoyl-N-hydroxysuccinimide ester), EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), DSS(disuccinimidylsuberate), HSAB (N-hydroxysuccinimidyl-4-azidobenzoate),and sulfo-SANPAH(sulfosuccinimidyl-6(4′-amido-2′-nitrophenylamido)hexanoate).
 9. Themethod of claim 8 wherein the intramolecularly, covalently cross-linkedprotein antigen is further modified to comprise a signal generatinggroup or a ligand for binding to a solid support.
 10. The method ofclaim 9 wherein the signal generating group is selected from the groupconsisting of enzymes, fluorescent groups and chemiluminescent groups.11. The method of claim 9 wherein the ligand is biotin or streptavidin.12. The method of claim 1 wherein the intramolecularly, covalentlycross-linked protein antigen is further modified to comprise a signalgenerating group or a ligand for binding to a solid support.
 13. Themethod of claim 1 wherein the intramolecularly, covalently cross-linkedprotein antigen is covalently linked to biotin or streptavidin.